Treatment of a variety of cultured human cells with Type I interferon (IFNs) induces a rapid increase in the rate of transcription of several cellular genes. This lab is interested in the mechanism by which IFNs are capable of activating and then subsequently inhibiting the expression of such genes. Phorbol ester-mediated inhibition of IFN-induced ISG54K expression: The following facts concerning the inhibitory effects of phorbol esters on IFN-activated ISG54K expression have been established. The actions of phorbol esters are reversed by inhibitors of protein synthesis. The mechanisms by which long term IFN treatment of cells down regulates ISG54 gene expression are distinct from those by which phorbol esters inhibit ISG54 expression. We have mapped two regions within the ISG54 gene that allow phorbol esters to repress IFN-activated gene expression. One is region at the cap site and one is the interferon-stimulated response element (ISRE). IFN-induced proteins that bind the ISRE are also inhibited by phorbol esters. We are presently purifying a protein which destroys the ISGF3 transcription complex from all PMA treated cells. We have recently developed a cell free system where IFNalpha can activate the formation of ISGF3 in vitro. This system has enabled us to demonstrate that the component of the ISGF3 transcription complex which is modified by IFNalpha (ISGF3alpha) is associated with the plasma membrane, and its activation involves a protein kinase. Using a combination of specific tyrosine kinase and phosphatase inhibitors, and monoclonal antiphosphotyrosine antibodies, we now are able to demonstrate that IFNalpha activated transcription involves at least a two step process where a tyrosine phosphatase and a tyrosine kinase lead to modification of ISGF3alpha and subsequent formation of the complete complex. The ISGF3 complex is specifically disrupted by protein tyrosine phosphatase and can be reversibly dissociated by the phosphotyrosine analogue phenylphosphate. This suggested that SH2 and/or SH3 domains may be required for the stable formation of this transcription complex.